An inhibitor of methionine aminopeptidase type-2, PPI-2458, ameliorates the pathophysiological disease processes of rheumatoid arthritis.

OBJECTIVE: To elucidate the role of methionine aminopeptidase type-2 (MetAP-2) in the clinical pathology of rheumatoid arthritis, arthritis was induced in rats by administration of peptidoglycan-polysaccharide (PG-PS). DESIGN: The inhibitor of MetAP-2, PPI-2458, was administered orally at 5 mg/kg every other day during 3 distinct phases of the disease. In vitro studies were performed to clarify in vivo findings. RESULTS: Ankle swelling was completely alleviated by MetAP-2 inhibition. Inhibition of MetAP-2 in blood and tissues correlated with protection against PG-PS-induced arthritis. Histopathology of the tarsal joints improved following PPI-2458 administration, including a significant improvement of bone structure. In in vitro studies, osteoclast formation and activity were inhibited by PPI-2458, a mechanism not previously attributed to MetAP-2 inhibition. CONCLUSIONS: The important role that MetAP-2 has in the pathophysiological disease processes of PG-PS arthritis provides a strong rationale for evaluating PPI-2458 as a disease modifying antirheumatic treatment for rheumatoid arthritis.

Prostanoid receptors: ontogeny and implications in vascular physiology.

Prostanoids exert significant effects on circulatory beds. They play a role in the response of the vasculature to adjustments in perfusion pressure and oxygen and carbon dioxide tension, and they mediate the actions of numerous factors. The role of prostanoids in governing circulation of the perinate is suggested to surpass that in the adult. Prostanoids are abundantly generated in the perinate. They have been implicated in autoregulation of blood flow as studied in brain and eyes. Prostaglandins are also dominant regulators of ductus arteriosus tone. The effects of these autacoids are mediated through specific G protein-coupled receptors. In addition to the pharmacological characterization of the prostanoid receptors, important advances in understanding the biology of these receptors have been made in the last decade. Their cloning and the development of animals with disrupted genes of these receptors have been very informative. The involvement of prostanoid receptors in the developing subject, especially on brain and ocular vasculature and on ductus arteriosus, has also begun to be investigated; the expression of these receptors changes with development. Some but not all of the ontogenic changes in these receptors are attributed to homologous regulation. Interestingly, in the process of elucidating their effects, functional perinuclear prostaglandin E2 receptors have been uncovered. This article reviews prostanoid receptors and addresses implications on the developing subject with attention to vascular physiology.

2,3-Dinor-5,6-dihydro-15-F(2t)-isoprostane: a bioactive prostanoid metabolite.

15-F(2t)-isoprostane (15-F(2t)-IsoP), also termed 8-isoprostaglandin F(2alpha), is one of a series of prostanoids formed by free radical-mediated peroxidation of arachidonic acid and exerts potent biological actions such as vasoconstriction. We recently demonstrated that 15-F(2t)-IsoP is metabolized in humans to a major metabolite, 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (15-F(2t)-IsoP-M). 15-F(2t)-IsoP-M can also potentially be formed as a product of free radical-induced oxidation of the low abundance fatty acid gamma-linolenic acid. We confirmed that 15-F(2t)-IsoP-M is generated during oxidation of gamma-linolenic acid and explored whether it may exhibit biological activity. 15-F(2t)-IsoP-M caused marked constriction of porcine surface retinal and intraparenchymal brain microvessels, comparable to that observed with 15-F(2t)-IsoP. These effects were associated with increased thromboxane A(2) (TXA(2)) formation and were virtually abolished by TXA(2)-synthase and -receptor inhibitors (CGS-12970 and L-670596). Vasoconstriction induced by either 15-F(2t)-IsoP or 15-F(2t)-IsoP-M on perfused ocular choroid was also abrogated by TXA(2)-synthase inhibition as well as by removal of endothelium. Similar to 15-F(2t)-IsoP, 15-F(2t)-IsoP-M evoked vasoconstriction and TXA(2) generation by activating Ca(2+) influx from nonvoltage-gated channels (SK&F96365 sensitive) in the retina and from both nonvoltage- and N-type voltage-gated Ca(2+) channels (omega-conotoxin MVIIA sensitive), respectively, in brain endothelial and astroglial cells; smooth muscle cells were unresponsive to both agents. Cross-desensitization experiments further suggest that 15-F(2t)-IsoP and 15-F(2t)-IsoP-M act on the same receptor mechanism. Findings reveal a novel concept by which a beta-oxidation metabolite of 15-F(2t)-IsoP that can also be formed by nonenzymatic oxidation of gamma-linolenic acid is equivalently bioactive to 15-F(2t)-IsoP and may prolong the vascular actions of F(2)-IsoPs.

Characterization of PGE2 receptors in fetal and newborn ductus arteriosus in the pig.

We compared the total density and the relative expression of EP receptor (EP) subtypes in ductus arteriosus (DA) of the newborn with that of the fetal piglet. Saturation binding experiments showed 3-fold less PGE2 receptors in the newborn than in the fetus because of loss of EP3 and EP4 receptors thus explaining, at least partly, the reduced responsiveness to PGE2 of the newborn DA. Displacement experiments showed that the relative proportions of EP2, EP3, and EP4 were similar in the fetal DA but only EP2 was detected in the DA of the newborn pig. Hence, PGE2 effects in the newborn DA seem to be exclusively mediated by EP2 receptors both in vitro and in vivo. These findings may help to propose more specific therapies for regulation of DA's tone in certain newborns for whom conventional therapy is contraindicated.

Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells.

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S1P activation of eNOS is approximately 10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(1179) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.

Permeability of endothelial monolayers to albumin is increased by bradykinin and inhibited by prostaglandins.

Using monolayers of bovine aortic endothelial cells (BAEC) in modified Boyden chambers, we examined the role of prostaglandins (PGs) in the bradykinin (BK)-induced increase of albumin permeability. BK induced a concentration-dependent increase of the permeability of BAEC, which reached 49.9 +/- 1% at the concentration of 10(-8) M. Two inhibitors of the prostaglandin G/H synthase, indomethacin (2.88 microM) and ibuprofen (10 microM), potentiated BK-induced permeability 1.8- and 3.9-fold, respectively. Exogenously administered PGE2 and iloprost, a stable analog of prostacyclin, attenuated the effect of BK in a concentration-dependent manner. Butaprost equally reduced the effect of BK, suggesting the participation of the EP2 receptor in this phenomenon. However, the EP4-selective antagonist AH-23848 did not significantly inhibit the protective effect of PGE2. The inhibitory effect of PGE2 was reversed by the adenylate cyclase inhibitor MDL-12330A (10 microM). These results suggest that BK-induced increase of permeability of BAEC monolayer to (125)I-labeled albumin is negatively regulated by PGs. This postulated autocrine activity of PGs may involve an increase in the intracellular level of cAMP.

Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase.

Activation of the bradykinin B2 receptor in endothelial cells initiates a complex array of cellular responses mediated by diverse signaling pathways, including stimulation of the mitogen-activated protein (MAP) kinase cascade and activation of the endothelial isoform of nitric-oxide synthase (eNOS). Several protein kinases have been implicated in eNOS regulation, but the role of MAP kinases remains less well understood. We explored the interactions between eNOS and components of the MAP kinase pathway in bovine aortic endothelial cells (BAEC). Using co-immunoprecipitation experiments, we isolated eNOS in a complex with the MAP kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as the protein kinases Raf-1 and Akt. Within minutes of adding bradykinin to BAEC, the eNOS-Raf-1-ERK-Akt heteromeric complex dissociated, and it subsequently reassociated following more prolonged agonist stimulation. Bradykinin treatment of BAEC led to the activation of ERK, associated with an increase in phosphorylation of eNOS; phosphorylation of eNOS by ERK in vitro significantly reduced eNOS enzyme activity. Evidence for the direct phosphorylation of eNOS by MAP kinase in BAEC came from "back-phosphorylation" experiments using [gamma-(32)P]ATP and ERK in vitro to phosphorylate eNOS isolated from cells previously treated with bradykinin or the MAP kinase inhibitor PD98059. The ERK-catalyzed in vitro (32)P phosphorylation of eNOS isolated from BAEC treated with bradykinin was significantly attenuated compared with untreated cells, indicating that bradykinin treatment led to the phosphorylation of ERK-sensitive sites in cells. Conversely, eNOS isolated from endothelial cells pretreated with the MAP kinase inhibitor PD98059 showed increased ERK-promoted phosphorylation in vitro. Taken together, our results suggest that bradykinin-induced activation of ERK leads to eNOS phosphorylation and enzyme inhibition, a process influenced by the reversible associations of members of the MAP kinase pathway with eNOS.

Adenosine induces cyclic-AMP formation and inhibits endothelin-1 production/secretion in guinea-pig tracheal epithelial cells through A(2B) adenosine receptors.

1. The adenosine receptor subtype mediating adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation and the effect of its activation on endothelin-1 (ET-1) secretion were studied in primary cultures of tracheal epithelial cells. 2. Adenosine analogues showed the following rank order of potency (pD(2) value) and intrinsic activity on the generation of cyclic AMP by tracheal epithelial cells: 5'-N-ethylcarboxyamidoadenosine (NECA, A(1)/A(2A)/A(2B), pD(2): 5.44+/-0.16)>adenosine (ADO, non selective, pD(2): 4.99+/-0. 09; 71+/-9% of NECA response) >/=2-Cl-adenosine (2CADO, non selective, pD(2): 4.72+/-0.14; 65+/-9% of NECA response)>>>CGS21680 (A(2A); inactive at up to 100 microM). 3. Cyclic AMP formation stimulated by NECA in guinea-pig tracheal epithelial cells was inhibited by adenosine receptor antagonist with the following order of apparent affinity (pA(2) value): Xanthine amine congeners (XAC, A(2A)/A(2B), 7.89+/-0.22)>CGS15943 (A(2A)/A(2B), 7.24+/-0. 26)>ZM241385 (A(2A), 6.69+/-0.14)>DPCPX (A(1), 6.51+/-0. 14)>3n-propylxanthine (weak A(2B), 4.30+/-0.10). This rank order of potency is typical for A(2B)-adenosine receptor. 4. Adenosine decreased basal and LPS-stimulated irET production in a concentration-dependent manner. Moreover, NECA but not CGS21680 inhibited LPS-induced irET production. 5. The inhibitory effect of NECA on LPS-induced irET production was reversed by XAC (pA(2)=8.84+/-0. 12) and DPCPX (pA(2)=8.10+/-0.22). 6. These results suggested that adenosine increased cyclic AMP formation and inhibited irET production/secretion by guinea-pig tracheal epithelial cells through the activation of a functional adenosine receptor that is most likely the A(2B) subtype. This adenosine receptor may be involved in the regulation of the level of ET-1 production/secretion by guinea-pig tracheal epithelial cells in physiological as well as in pathophysiological conditions.

N- and C-terminal structure-activity study of angiotensin II on the angiotensin AT2 receptor.

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV >> Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.

Characterization of AT4 receptor from bovine aortic endothelium with photosensitive analogues of angiotensin IV.

Newly developed photosensitive analogues of AngIV were used to characterize the AT4 receptor of bovine aortic endothelial cells. The photoactivatable AngIV analogues [N3-Phe6]AngIV and [Bpa6]AngIV displayed high affinities for AT4 receptor, with IC50's of 3.7 +/- 0.3 and 19.1 +/- 3.5 nM, respectively. The radioiodinated ligands showed a good efficiency of photoaffinity labeling demonstrated by high proportions (60-75%) of acid-resistant binding. Covalently labeled receptor was solubilized under reducing or nonreducing conditions and subjected to SDS-PAGE. Under nonreducing conditions, autoradiographies revealed a major band of Mr 186 +/- 2 kDa and a minor band of Mr 241 +/- 6 kDa. The labeling of these bands was completely abolished in the presence of 10 microM AngIV. Under reducing conditions, only the low Mr 186 kDa band was revealed. After endoglycosidase digestion with an enzyme that cleaves N-linked saccharides, the Mr of the denatured AT4 receptor was decreased by 31% to a value of 129 +/- 10 kDa. Kinetic studies revealed a stepwise process of AT4 receptor deglycosylation by endoglycosidase F, suggesting at least two different sites of N-linked saccharides. Mild trypsin treatment of photolabeled endothelial cell membranes released a large fragment of Mr 177 +/- 3 kDa which accounts for about 95% of the whole receptor molecular mass. These results demonstrate that [N3-Phe6]AngIV and [Bpa6]AngIV are very efficient tools for selective photoaffinity labeling of AT4 receptor. We have shown that AT4 receptor is a 186 kDa integral membrane glycoprotein with a very large extracellular domain. These properties are consistent with those of a growth factor or cytokine receptor.

Study on the functionality and molecular properties of the AT4 receptor.

Whereas the role of angiotensin II (Ang II) has been clarified in numerous tissues and cell types, the physiological relevance of its C-terminal (3-8) degradation fragment, angiotensin IV (Ang IV), is unclear. Previously, we characterized a specific binding site for Ang IV in the bovine adrenal cortex and on bovine aortic endothelial cells (BAEC). In the present study, we tried to assess the functionality and mechanism of action of this receptor for Ang IV (AT4 receptor). Our results revealed that none of the classical second messengers (i.e., cAMP, Ca2+, inositol phosphates, nitric oxide or arachidonic acid derivatives) was modified significantly during acute (less than 1 h) stimulation of cells with Ang IV. Under normal culture conditions, BAEC efficiently internalized 125I-Ang IV. After a 2 h incubation at 37 degrees C, acid-resistant binding corresponded to about 50% of total cell-associated radioactivity. This rapid internalization process suggests that the AT4 receptor is a functional protein. With a photoaffinity labeling approach, we revealed some properties of the AT4 receptor that are consistent with those of a growth factor or cytokine receptor.

Desensitization of AT1 receptor-mediated cellular responses requires long term receptor down-regulation in bovine adrenal glomerulosa cells.

Angiotensin II (Ang II) regulates aldosterone production in bovine adrenal glomerulosa cells by interacting with the AT1 receptor. This receptor is coupled to a G protein that controls the activity of phospholipase C. With a primary culture of bovine adrenal glomerulosa cells, we evaluated the desensitization of cellular responses after pretreatment with Ang II. When cells were pretreated for 30 min with 1 microM Ang II at 37 C, we observed a 48% loss of [125I]Ang II-binding activity. Scatchard analysis revealed that this decreased binding activity corresponded to a 53% loss of the total number of binding sites. This phenomenon was time dependent, with a t(1/2) of 20 min, and a maximal loss of 76% of the total binding sites was observed after 14 h. A time-dependent decrease in AT1 receptor messenger RNA levels was also observed after pretreatment with 1 microM Ang II for 12-24 h. Taken together, these results are interpreted as a down-regulation of the AT1 receptor. Desensitization of phospholipase C activity under similar conditions was, however, a slower process, with a t(1/2) of 9 h and a maximal response reduction of 83% observed after 24 h. Dose-response experiments indicated that maximal phospholipase C desensitization was obtained in the presence of 1 microM Ang II, with an EC50 of 90 nM. The desensitization was of a homologous nature, as a 24-h pretreatment with Ang II did not affect bradykinin-induced inositol phosphate production. A 24-h pretreatment with 1 microM Ang II also significantly desensitized the steroidogenic effect of Ang II and the potentiating effect of Ang II on ACTH-induced cAMP production. Lower concentrations of Ang II (10 nM) did not produce any desensitizing effect on these two parameters. This study provides evidence that glomerulosa cells are functionally resistant to short term desensitization of the AT1 receptor and that long term down-regulation with high concentrations of Ang II is needed to desensitize AT1-mediated cellular responses.

Mechanism of action of leukotriene D4 on guinea pig tracheal smooth muscle cells: roles of Ca++ influx and intracellular Ca++ release.

The effects of leukotriene D4 (LTD4) on the concentration of intracellular cytosolic free calcium ([Ca++]i) and on phosphoinositide hydrolysis were studied in cultured guinea pig tracheal smooth muscle cells. In Fura-2-loaded cells, LTD4 (10(-9)-10(-6) M) induced concentration-dependent changes in [Ca++]i consisting of a slow, transient increase followed by a sustained phase. Preincubation of cells with LTD4 receptor antagonist MK-571 (10(-6) M) blocked the increase in [Ca++]i. Similarly, LTD4-induced inositol phosphate ([3H]InsP(s) synthesis was transient, concentration-dependent and inhibited by the LTD4 antagonist. In the absence of extracellular Ca++, LTD4 failed to induce [Ca++]i increases and [3H]InsP(s) formation. Accordingly, NiCl2 completely inhibited the LTD4-stimulated [3H]InsP(s) synthesis. Nifedipine (10(-5) M) had a slight inhibitory effect on [Ca++]i increase but significantly reduced (40-50%) the [3H]lnsP(s) accumulation. These findings indicate that LTD4-stimulated inositol phosphate synthesis and [Ca++]i increases in tracheal smooth muscle cells are receptor-mediated events and are dependent on the availability of extracellular Ca++. It is suggested that Ca++ influx plays a major role in the LTD4 signal transduction mechanism.

Calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells.

We provided evidence that calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells. In cells labeled for 16 hr with 3H-arachidonic acid, ionomycin and Ca2(+)-mobilizing hormones such as bradykinin, thrombin and platelet activating factor induced arachidonic acid release. However, arachidonic acid release was not induced by agents known to increase cyclic AMP (forskolin, isoproterenol) or cyclic GMP (sodium nitroprusside). Bradykinin induced the release of arachidonic acid in a dose-dependent manner (EC50 = 1.6 +/- 0.7 nM). This increase was rapid, reaching a maximal value of fourfold above basal level in 15 min. In a Ca2(+)-free medium, bradykinin was still able to release arachidonic acid but with a lower efficiency. Quinacrine (300 microM), a blocker of PLA2, completely inhibited bradykinin-induced arachidonic acid release. The B2 bradykinin receptor antagonist HOE-140 completely inhibited bradykinin-induced arachidonic acid release. The B1-selective agonist DesArg9-bradykinin was inactive and the B1-selective antagonist [Leu8] DesArg9-bradykinin had no significant effect on bradykinin-induced arachidonic acid release. The phospholipase C inhibitor U-73122 (100 microM) decreased bradykinin-induced arachidonic acid release. The calmodulin inhibitor W-7 (50 microM) drastically reduced the bradykinin- and ionomycin-induced arachidonic acid release. Also, forskolin decreased bradykinin-induced arachidonic acid release. These results suggest that the activation of PLA2 by bradykinin in BAEC is a direct consequence of phospholipase C activation. Ca2(+)-calmodulin appears to be the prominent activator of PLA2 in this system.

Characterization of a binding site for angiotensin IV on bovine aortic endothelial cells.

We have characterized a specific binding site for angiotensin IV on bovine aortic endothelial cell membranes. Pseudo-equilibrium studies at 37 degrees C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (Kd = 0.71; average of two experiments that yielded values of 0.71 and 0.72 nM). The binding site is saturable and relatively abundant with a maximal binding capacity of 0.59 pmol/mg protein (average of two experiments that yielded values of 0.39 and 0.78 pmol/mg of protein). Non-equilibrium kinetic analyses at 37 degree C revealed a calculated Kd of 59 pM (average of two experiments that yielded values of 67 and 50 pM). The binding site displays a high affinity for angiotensin receptors AT1 or AT2. An analysis of specificity showed that the binding site displays a high affinity for angiotensin IV, low affinities for angiotensin II, [Sar1, Val5, Ala8]angiotensin II and does not recognize L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'-(1 H-tetrazole-5-yl)[1,1'-biphenyl]-4-yl)methyl]-3H-imidazo[4, 5-beta]pyridine H2O) and PD 123319 (1-[4-dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl) 4,5,6,7-tetrahydro-1 H-imidazo[4,5-c]pyridine-6-carboxylic acid). A few unrelated hormones (bradykinin, [Arg8] vasopressin, endothelin-1, atrial natriuretic factor, isoproterenol and adrenocorticotropic hormone) were unable to inhibit any 125I-angiotensin IV binding. The affinities of different structural analogues of angiotensin IV revealed that the N-terminal position is critical for receptor recognition and the C-terminal proline is also important. GTP gamma S and polyvinyl sulfate did not affect the binding, suggesting that the receptor is not coupled to a G-protein. The divalent cations Mg2+ and Ca2+ were shown to diminish the binding of 125I-angiotensin IV. Cross-linking of 125I-angiotensin IV to bovine aortic endothelial cell membranes in the presence of disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a major band of 186 +/- 12 kDa. The presence in high concentration of this angiotensin binding site on aortic endothelial cells suggest the existence of a novel mechanism involved in the control of vascular tone or vascular permeability.

A specific binding site recognizing a fragment of angiotensin II in bovine adrenal cortex membranes.

We have characterized a specific binding site for angiotensin IV in bovine adrenal cortex membranes. Pseudo-equilibrium studies at 37 degrees C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (Kd = 0.24 +/- 0.03 nM). The binding site is saturable and relatively abundant (maximal binding capacity around 0.5 pmol/mg protein). Non-equilibrium kinetic analyses at 37 degrees C revealed a calculated kinetic Kd of 47 pM. The binding site is pharmacologically distinct from the classic angiotensin receptors AT1 or AT2. Competitive binding studies with bovine adrenal cortex membranes demonstrated the following rank order of effectiveness: angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) = angiotensin II-(3-7) (Val-Tyr-Ile-His-Pro) > angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) > or = angiotensin II-(4-7) (Tyr-Ile-His-Pro) > angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > angiotensin II-(1-6) (Asp-Arg-Val-Tyr-Ile-His) > angiotensin II-(4-8) (Tyr-Ile-His-Pro-Phe) > > > angiotensin II-(3-6) (Val-Tyr-Ile-His), angiotensin II-(4-6) (Tyr-Ile-His), L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'(1-H-tetrazol-5-yl)[1,1'-biphenyl]-4-y l) methyl]-3-H-imidazo[4,5-beta]pyridine H2O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl)4,5,6 ,7- tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid). The divalent cations Mg2+ and Ca2+ were shown to diminish the binding of 125I-angiotensioffn IV to bovine adrenal cortex membranes.(ABSTRACT TRUNCATED AT 250 WORDS)